A Risk Assessment Framework for Seed Degeneration: Informing an Integrated Seed Health Strategy for Vegetatively Propagated Crops.

Pathogen buildup in vegetative planting material, termed seed degeneration, is a major problem in many low-income countries. When smallholder farmers use seed produced on-farm or acquired outside certified programs, it is often infected. We introduce a risk assessment framework for seed degeneration, evaluating the relative performance of individual and combined components of an integrated seed health strategy. The frequency distribution of management performance outcomes was evaluated for models incorporating biological and environmental heterogeneity, with the following results. (1) On-farm seed selection can perform as well as certified seed, if the rate of success in selecting healthy plants for seed production is high; (2) when choosing among within-season management strategies, external inoculum can determine the relative usefulness of 'incidence-altering management' (affecting the proportion of diseased plants/seeds) and 'rate-altering management' (affecting the rate of disease transmission in the field); (3) under severe disease scenarios, where it is difficult to implement management components at high levels of effectiveness, combining management components can be synergistic and keep seed degeneration below a threshold; (4) combining management components can also close the yield gap between average and worst-case scenarios. We also illustrate the potential for expert elicitation to provide parameter estimates when empirical data are unavailable. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Pea seed-borne mosaic virus Pathosystem Drivers under Mediterranean-Type Climatic Conditions: Deductions from 23 Epidemic Scenarios.

Drivers of Pea seed-borne mosaic virus (PSbMV) epidemics in rainfed field pea crops were examined under autumn to spring growing conditions in a Mediterranean-type environment. To collect aphid occurrence and PSbMV epidemic data under a diverse range of conditions, 23 field pea data collection blocks were set up over a 6-year period (2010 to 2015) at five locations in the southwest Australian grain-growing region. PSbMV infection levels in seed sown (0.1 to 13%), time of sowing (22 May to 22 June), and cultivar (Kaspa or PBA Twilight) varied with location and year. Throughout each growing season, rainfall data were collected, leaf and seed samples were tested to monitor PSbMV incidence in the crop and transmission from harvested seed, and sticky traps were used to monitor flying aphid numbers. Winged migrant Acyrthosiphon kondoi, Lipaphis erysimi, Myzus persicae, and Rhopalosiphum padi were identified in green tile traps in 2014 and 2015. However, no aphid colonization of field pea plants ever occurred in the blocks. The deductions made from collection block data illustrated how the magnitude of PSbMV spread prior to flowering is determined by two primary epidemic drivers: (i) PSbMV infection incidence in the seed sown, which defines the magnitude of virus inoculum source for within-crop spread by aphids, and (ii) presowing rainfall that promotes background vegetation growth which, in turn, drives early-season aphid populations and the time of first arrival of their winged migrants to field pea crops. Likely secondary epidemic drivers included wind-mediated PSbMV plant-to-plant contact transmission and time of sowing. PSbMV incidence at flowering time strongly influenced transmission rate from harvested seed to seedlings. The data collected are well suited for development and validation of a forecasting model that informs a Decision Support System for PSbMV control in field pea crops.

Pea seed-borne mosaic virus: Stability and Wind-Mediated Contact Transmission in Field Pea.

Pea seed-borne mosaic virus (PSbMV) stability in sap and its contact transmission between field pea plants were investigated in glasshouse experiments. When infective leaf sap was kept at room temperature and inoculated to plants in the absence of abrasive, it was still highly infective after 6 h and low levels of infectivity remained after 30 h. PSbMV was transmitted from infected to healthy plants by direct contact when leaves were rubbed against each other. It was also transmitted when intertwining healthy and PSbMV-infected plants were blown by a fan to simulate wind. When air was blown on plants kept at 14 to 20°C, contact transmission of PSbMV occurred consistently and the extent of transmission was enhanced when plants were dusted with diatomaceous earth prior to blowing. In contrast, when plants were kept at 20 to 30°C, blowing rarely resulted in transmission. No passive contact transmission occurred when healthy and infected plants were allowed to intertwine together. This study demonstrates that PSbMV has the potential to be transmitted by contact when wind-mediated wounding occurs in the field. This may play an important role in the epidemiology of the virus in field pea crops, especially in situations where contact transmission expands initial crop infection foci before aphid arrival.

Pea seed-borne mosaic virus in Field Pea: Widespread Infection, Genetic Diversity, and Resistance Gene Effectiveness.

From 2013 to 2015, incidences of Pea seed-borne mosaic virus (PSbMV) infection were determined in semi-leafless field pea (Pisum sativum) crops and trial plots growing in the Mediterranean-type environment of southwest Australia. PSbMV was found at incidences of 2 to 51% in 9 of 13 crops, 1 to 100% in 20 of 24 cultivar plots, and 1 to 57% in 14 of 21 breeding line plots. Crops and plots of 'PBA Gunyah', 'Kaspa', and 'PBA Twilight' were frequently PSbMV infected but none of PSbMV resistance gene sbm1-carrying 'PBA Wharton' plants were infected. In 2015, 14 new PSbMV isolates obtained from these various sources were sequenced and their partial coat protein (CP) nucleotide sequences analyzed. Sequence identities and phylogenetic comparison with 39 other PSbMV partial CP nucleotide sequences from GenBank demonstrated that at least three PSbMV introductions have occurred to the region, one of which was previously unknown. When plants of 'Greenfeast' and PBA Gunyah pea (which both carry resistance gene sbm2) and PBA Wharton and 'Yarrum' (which carry sbm1) were inoculated with PSbMV pathotype P-2 isolate W1, resistance was overcome in a small proportion of plants of each cultivar, showing that resistance-breaking variants were likely to be present. An improved management effort by pea breeders, advisors, and growers is required to diminish infection of seed stocks, avoid sbm gene resistance being overcome in the field, and mitigate the impact of PSbMV on seed yield and quality. A similar management effort is likely to be needed in field pea production elsewhere in the world.

Potato virus Y: Contact Transmission, Stability, Inactivation, and Infection Sources.

In glasshouse experiments, two isolates of Potato virus Y 'O' strain (PVYO) were transmitted from infected to healthy potato plants by direct contact when leaves were rubbed against each other, when cut surfaces of infected tubers were rubbed onto leaves, and to a limited extent, when blades contaminated with infective sap were used to cut healthy potato tubers. However, no tuber-to-tuber transmission occurred when blades were used to cut healthy tubers after cutting infected tubers. When leaf sap from potato plants infected with two PVYO isolates was kept at room temperature, it was highly infective for 6 to 7 h and remained infectious for up to 28 h. Also, when sap from infected leaves with one isolate was applied to five surfaces (cotton, hessian, metal, rubber vehicle tire, and wood) and left to dry for up to 24 h before each surface was rubbed onto healthy tobacco plants, PVYO remained infective for 24 h on tire and metal, 6 h on cotton and hessian, and 3 h on wood. The effectiveness of disinfectants at inactivating this isolate was evaluated by adding them to sap from infected leaves which was then rubbed onto healthy tobacco plants. None of the plants became infected when bleach (42 g/liter sodium hypochlorite, diluted 1:4) or Virkon-S (potassium peroxymonosulfate 50% wt/wt, diluted to 1%) was used. A trace of infection remained after using nonfat milk powder (20% wt/vol). PVY infection sources were studied in 2011-2012 in the main potato growing regions of southwest Australia. In tests on >17,000 potato leaf samples, PVY was detected at low levels in seed (4/155) and ware (6/51) crops. It was also detected in volunteer potatoes from a site with a previous history of PVY infection in a seed crop. None of the 15 weed species tested were PVY infected. Plants of Solanum nigrum were symptomlessly infected with PVYO after sap inoculation, and no seed transmission was detected (>2,500 seeds). This study demonstrates PVYO can be transmitted by contact and highlights the need to include removal of volunteer potatoes and other on-farm hygiene practices (decontaminating tools, machinery, clothing, etc.) in integrated disease management strategies for PVY in potato crops.

Potato spindle tuber viroid: Stability on Common Surfaces and Inactivation With Disinfectants.

The length of time Potato spindle tuber viroid (PSTVd) remained infective in extracted tomato leaf sap on common surfaces and the effectiveness of disinfectants against it were investigated. When sap from PSTVd-infected tomato leaves was applied to eight common surfaces (cotton, wood, rubber tire, leather, metal, plastic, human skin, and string) and left for various periods of time (5 min to 24 h) before rehydrating the surface and rubbing onto healthy tomato plants, PSTVd remained infective for 24 h on all surfaces except human skin. It survived best on leather, plastic, and string. It survived less well after 6 h on wood, cotton, and rubber and after 60 min on metal. On human skin, PSTVd remained infective for only 30 min. In general, rubbing surfaces contaminated with dried infective sap directly onto leaves caused less infection than when the sap was rehydrated with distilled water but overall results were similar. The effectiveness of five disinfectant agents at inactivating PSTVd in sap extracts was investigated by adding them to sap from PSTVd-infected leaves before rubbing the treated sap onto leaves of healthy tomato plants. Of the disinfectants tested, 20% nonfat dried skim milk and a 1:4 dilution of household bleach (active ingredient sodium hypochlorite) were the most effective at inactivating PSTVd infectivity in infective sap. When reverse-transcription polymerase chain reaction was used to test the activity of the five disinfectants against PSTVd in infective sap, it detected PSTVd in all instances except in sap treated with 20% nonfat dried skim milk. This study highlights the stability of PSTVd in infective sap and the critical importance of utilizing hygiene practices such as decontamination of clothing, tools, and machinery, along with other control measures, to ensure effective management of PSTVd and, wherever possible, its elimination in solanaceous crops.

Polymyxa graminis Isolates from Australia: Identification in Wheat Roots and Soil, Molecular Characterization, and Wide Genetic Diversity.

Polymyxa graminis is an obligate parasite of roots and an important vector of viruses that damage cereal crops in different parts of the world. In 2011 and 2012, P. graminis was identified infecting 11 wheat root samples from three widely dispersed locations in southwest Australia. Its presence was detected by polymerase chain reaction (PCR) and confirmed by DNA sequencing of the transcribed regions of its ribosomal RNA genes (rDNA) and observing sporosori of characteristic morphology and size in stained wheat roots. Also, when soil samples were collected from two locations where P. graminis was found and wheat bait plants grown in them, P. graminis was detected in their roots by PCR. Ribosomal DNA sequences of six southwest Australian isolates were obtained from wheat roots, and one northeast Australian isolate from barley roots. When these seven P. graminis sequences were compared with others from GenBank by phylogenetic analysis, three southwest Australian isolates were classified as P. graminis f. sp. temperata (ribotypes Ia and Ib), and three as f. sp. tepida (ribotypes IIa and IIb). P. graminis f. sp. temperata and tepida both occur in temperate growing regions of other continents and are associated with transmission of soil-borne viruses to cereal crops. The P. graminis isolate from northeast Australia was sufficiently distinct from the five existing sequence groups for it to be placed into a newly proposed grouping, ribotype VI, which also included an isolate from tropical West Africa. However, when randomly collected wheat leaf samples from 39 field crops from 27 widely dispersed locations, 21 individual wheat plant samples collected from low lying areas within 21 fields at 11 locations, and wheat bait plants growing in five soil samples from two locations were tested by reverse transcription (RT)-PCR for the presence of Soil-borne wheat mosaic virus, Soil-borne cereal mosaic virus, Wheat spindle streak mosaic virus, and furoviruses in general, no virus infection was detected. These findings suggest at least three P. graminis introductions into Australia, and the occurrence of f. sp. temperata (ribotype I) and f. sp. tepida (ribotype II) suggests that, if not already present, soil-borne cereal viruses are likely to become established should they become introduced to the continent in the future.

Black Pod Syndrome of Lupinus angustifolius Is Caused by Late Infection with Bean yellow mosaic virus.

Black pod syndrome (BPS) causes devastating losses in Lupinus angustifolius (narrow-leafed lupin) crops in Australia, and infection with Bean yellow mosaic virus (BYMV) was suggested as a possible cause. In 2011, an end-of-growing-season survey in which L. angustifolius plants with BPS were collected from six locations in southwestern Australia was done. Tissue samples from different positions on each of these symptomatic plants were tested for BYMV and generic potyvirus by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR). Detection was most reliable when RT-PCR with generic potyvirus primers was used on tissue taken from the main stem of the plant just below the black pods. Partial coat protein nucleotide sequences from eight isolates from BPS-symptomatic L. angustifolius plants all belonged to the BYMV general phylogenetic group. An initial glasshouse experiment revealed that mechanical inoculation of L. angustifolius plants with BYMV after pods had formed caused pods to turn black. This did not occur when the plants were inoculated before this growth stage (at first flowering) because BYMV infection caused plant death. A subsequent experiment in which plants were inoculated at eight different growth stages confirmed that BPS was only induced when L. angustifolius plants were inoculated after first flowering, when pods had formed. Thus, BYMV was isolated from symptomatic L. angustifolius survey samples, inoculated to and maintained in culture hosts, inoculated to healthy L. angustifolius test plants inducing BPS, and then successfully reisolated from them. As such, Koch's postulates were fulfilled for the hypothesis that late infection with BYMV causes BPS in L. angustifolius plants.

First Report of Wheat mosaic virus Infecting Wheat in Western Australia.

In eastern Australia, there have been several as yet unconfirmed reports of Wheat mosaic virus (WMoV) infecting wheat (3). WMoV, previously known as High plains virus (HPV), is transmitted by the wheat curl mite (WCM, Aceria tosichella). It is often found in mixed infections with Wheat streak mosaic virus (WSMV), also transmitted by WCM (2,3). WSMV was first identified in Australia in 2003 (3). In October 2012, stunted wheat plants with severe yellow leaf streaking were common in a field experiment near Corrigin in Western Australia consisting of nine wheat cultivars. These symptoms were also common in two commercial crops of wheat cv. Mace near Kulin. Leaf samples (one per plant) from each location were tested by ELISA using specific antiserum to WMoV (syn. HPV 17200, Agdia, Elkhart, IN). At the field experiment, 20 leaf samples were collected at random from each wheat plot (4 replicates) and tested individually by ELISA. WMoV incidence was 5% for cv. Yipti, 16% for cvs Emu Rock, Wyalkatchem and Mace, 22% for cvs. Corack, Fortune, Calingiri, and Magenta, and 55% for cv. Cobra. From the two commercial wheat crops, 100 leaf samples were collected at random from each and tested by ELISA. WMoV incidence was 2 and 4%. In addition, 50 leaf samples of Hordeum leporinum (barley grass) and 20 of Lolium rigidum (annual ryegrass) were collected and tested by ELISA. WMoV incidence was 2% in H. leporinum, but 0% in L. rigidum. Infected H. leporinum plants were symptomless. Symptomatic wheat leaf samples from both sites were tested by RT-PCR using WMoV specific primers designed from its RNA3 sequence (1). The PCR products (339 bp) were sequenced and lodged in GenBank (Accession Nos KC337341 and KC337342). WMoV isolates from Corrigin (WA-CG12) and Kulin (WA-KU12) had identical sequences. When the nucleic acid sequences of WA-CG12 and WA-KU12 were compared with those of the three other WMoV isolates on GenBank, they had 100% nucleotide sequence identity with a Nebraska isolate (U60141), and 99.7% identity to two United States sweet corn isolates (AY836524 and AY836525). Ten symptomatic wheat plants were collected from each location, transplanted into pots and leaf samples tested individually for WMoV and WSMV (07048, Loewe, Germany) by ELISA. All were infected with both viruses and infested with WCM. WCM-infested glumes (>10 WCM/glume) were placed on the leaf sheaths of 60 wheat plants cv. Calingiri (35 with WA-CG12 and 25 with WA-KU12) and 13 sweet corn plants cv. Snow Gold (WA-CG12 only). In addition, 20 wheat and 10 sweet corn plants were left without infested glumes to be uninoculated controls. All 60 WCM-inoculated wheat plants became stunted with severe leaf streaking. When leaf samples from each plant were tested by ELISA 18 to 30 days later, both viruses were detected. WMoV was detected in all 13 WCM-inoculated sweet corn plants and WSMV in two of them. Plants with WMoV alone initially had short chlorotic leaf streaks that subsequently combined, causing broad streaks. These are typical WMoV symptoms for sweet corn (1). No symptoms developed and no virus was detected in any of the uninoculated wheat or sweet corn control plants. The WMoV nucleotide sequence obtained from an infected sweet corn plant was identical to those of WA-CG12 and WA-KU12. To our knowledge, this is the first confirmed report of WMoV presence in Australia. References: (1) B. S. M. Lebas et al. Plant Dis. 89:1103, 2005. (2) D. Navia et al. Exp. Appl. Acarol. 59:95, 2013. (3) J. M. Skare et al. Virology 347:343, 2006.

Zucchini yellow mosaic virus: Contact Transmission, Stability on Surfaces, and Inactivation with Disinfectants.

In glasshouse experiments, Zucchini yellow mosaic virus (ZYMV) was transmitted from infected to healthy zucchini (Cucurbita pepo) plants by direct contact when leaves were rubbed against each other, crushed, or trampled, and, to a lesser extent, on ZYMV-contaminated blades. When sap from zucchini plants infected with three ZYMV isolates was kept at room temperature for up to 6 h, it infected healthy plants readily. Also, when sap from ZYMV-infected leaves was applied to seven surfaces (cotton, plastic, leather, metal, rubber vehicle tire, rubber-soled footwear, and human skin) and left for up to 48 h before the ZYMV-contaminated surface was rubbed onto healthy zucchini plants, ZYMV remained infective for 48 h on tire, 24 h on plastic and leather, and up to 6 h on cotton, metal, and footwear. On human skin, ZYMV remained infective for 5 min only. The effectiveness of 13 disinfectants at inactivating ZYMV was evaluated by adding them to sap from ZYMV-infected leaves which was then rubbed on to healthy zucchini plants. None of the plants became infected when nonfat dried milk (20%, wt/vol) or bleach (sodium hypochlorite at 42 g/liter, diluted 1:4) were used. When ZYMV-infected pumpkin leaves were trampled by footwear and then used to trample healthy plants, all plants became infected; however, when contaminated footwear was dipped in a footbath containing bleach (sodium hypochlorite at 42 g/liter, diluted 1:4) before trampling, none became infected. This study demonstrates that ZYMV can be transmitted by contact and highlights the need for on-farm hygiene practices (decontaminating tools, machinery, clothing, and so on) to be included in integrated disease management strategies for ZYMV in cucurbit crops.

First Report of Alfalfa mosaic virus Infecting Tedera (Bituminaria bituminosa (L.) C.H. Stirton var. albomarginata and crassiuscula) in Australia.

Tedera (Bituminaria bituminosa (L.) C.H. Stirton vars albomarginata and crassiuscula) is being established as a perennial pasture legume in southwest Australia because of its drought tolerance and ability to persist well during the dry summer and autumn period. Calico (bright yellow mosaic) leaf symptoms occurred on occasional tedera plants growing in genetic evaluation plots containing spaced plants at Newdegate in 2007 and Buntine in 2010. Alfalfa mosaic virus (AlMV) infection was suspected as it often causes calico in infected plants (1,2) and infects perennial pasture legumes in local pastures (1,3). Because AlMV frequently infects Medicago sativa (alfalfa) in Australia and its seed stocks are commonly infected (1,3), M. sativa buffer rows were likely sources for spread by aphids to healthy tedera plants. When leaf samples from plants with typical calico symptoms from Newdegate (2007) and Buntine (2010) were tested by ELISA using poyclonal antisera to AlMV, Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV), only AlMV was detected. When leaf samples from 864 asymptomatic spaced plants belonging to 34 tedera accessions growing at Newdegate and Mount Barker in 2010 were tested by ELISA, no AlMV, BYMV, or CMV were detected, despite presence of M. sativa buffer rows. A culture of AlMV isolate EW was maintained by serial planting of infected seed of M. polymorpha L. (burr medic) and selecting seed-infected seedlings (1,3). Ten plants each of 61 accessions from the local tedera breeding program were grown at 20°C in an insect-proof air conditioned glasshouse. They were inoculated by rubbing leaves with infective sap containing AlMV-EW or healthy sap (five plants each) using Celite abrasive. Inoculations were always done two to three times to the same plants. When both inoculated and tip leaf samples from each plant were tested by ELISA, AlMV was detected in 52 of 305 AlMV-inoculated plants belonging to 36 of 61 accessions. Inoculated leaves developed local necrotic or chlorotic spots or blotches, or symptomless infection. Systemic invasion was detected in 20 plants from 12 accessions. Koch's postulates were fulfilled in 12 plants from nine accessions (1 to 2 of 5 plants each), obvious calico symptoms developing in uninoculated leaves, and AlMV being detected in symptomatic samples by ELISA, inoculation of sap to diagnostic indicator hosts (2) and RT-PCR with AlMV CP gene primers. Direct RT-PCR products were sequenced and lodged in GenBank. When complete nucleotide CP sequences (666 nt) of two isolates from symptomatic tedera samples and two from alfalfa (Aq-JX112758, Hu-JX112759) were compared with that of AlMV-EW, those from tedera and EW were identical (JX112757) but had 99.1 to 99.2% identities to the alfalfa isolates. JX112757 had 99.4% identity with Italian tomato isolate Y09110. Systemically infected tedera foliage sometimes also developed vein clearing, mosaic, necrotic spotting, leaf deformation, leaf downcurling, or chlorosis. Later-formed leaves sometimes recovered, but plant growth was often stunted. No infection was detected in the 305 plants inoculated with healthy sap. To our knowledge, this is the first report of AlMV infecting tedera in Australia or elsewhere. References: (1) B. A. Coutts and R. A. C. Jones. Ann. Appl. Biol. 140:37, 2002. (2) E. M. J. Jaspars and L. Bos. Association of Applied Biologists, Descriptions of Plant Viruses No. 229, 1980. (3) R. A. C. Jones. Aust. J. Agric. Res. 55:757, 2004.

Detection of Viruses in Sweetpotato from Honduras and Guatemala Augmented by Deep-Sequencing of Small-RNAs.

Sweetpotato (Ipomoea batatas) plants become infected with over 30 RNA or DNA viruses in different parts of the world but little is known about viruses infecting sweetpotato crops in Central America, the center of sweetpotato domestication. Small-RNA deep-sequencing (SRDS) analysis was used to detect viruses in sweetpotato in Honduras and Guatemala, which detected Sweet potato feathery mottle virus strain RC and Sweet potato virus C (Potyvirus spp.), Sweet potato chlorotic stunt virus strain WA (SPCSV-WA; Crinivirus sp.), Sweet potato leaf curl Georgia virus (Begomovirus sp.), and Sweet potato pakakuy virus strain B (synonym: Sweet potato badnavirus B). Results were confirmed by polymerase chain reaction and sequencing of the amplicons. Four viruses were detected in a sweetpotato sample from the Galapagos Islands. Serological assays available to two of the five viruses gave results consistent with those obtained by SRDS, and were negative for six additional sweetpotato viruses tested. Plants coinfected with SPCSV-WA and one to two other viruses displayed severe foliar symptoms of epinasty and leaf malformation, purpling, vein banding, or chlorosis. The results suggest that SRDS is suitable for use as a universal, robust, and reliable method for detection of plant viruses, and especially useful for determining virus infections in crops infected with a wide range of unrelated viruses.

Genetic variability of the coat protein sequence of pea seed-borne mosaic virus isolates and the current relationship between phylogenetic placement and resistance groups.

Nucleotide sequences of complete or partial coat protein (CP) genes were determined for 11 isolates of pea seed-borne mosaic virus (PSbMV) from Australia and one from China, and compared with known sequences of 20 other isolates. On phylogenetic analysis, the isolates from Australia and China grouped into 2 of 3 clades. Clade A contained three sub-clades (Ai, Aii and Aiii), Australian isolates were in Ai or Aiii, and the Chinese isolate in Aii. Clade A contained isolates in pathotypes P-1, P-2 and U-2; clade B, one isolate in P-2; and clade C, only isolates in P-4.

Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences.

Between 2006 and 2010, 5324 samples from at least 34 weed, two cultivated legume and 11 native species were collected from three cucurbit-growing areas in tropical or subtropical Western Australia. Two new alternative hosts of zucchini yellow mosaic virus (ZYMV) were identified, the Australian native cucurbit Cucumis maderaspatanus, and the naturalised legume species Rhyncosia minima. Low-level (0.7%) seed transmission of ZYMV was found in seedlings grown from seed collected from zucchini (Cucurbita pepo) fruit infected with isolate Cvn-1. Seed transmission was absent in >9500 pumpkin (C. maxima and C. moschata) seedlings from fruit infected with isolate Knx-1. Leaf samples from symptomatic cucurbit plants collected from fields in five cucurbit-growing areas in four Australian states were tested for the presence of ZYMV. When 42 complete coat protein (CP) nucleotide (nt) sequences from the new ZYMV isolates obtained were compared to those of 101 complete CP nt sequences from five other continents, phylogenetic analysis of the 143 ZYMV sequences revealed three distinct groups (A, B and C), with four subgroups in A (I-IV) and two in B (I-II). The new Australian sequences grouped according to collection location, fitting within A-I, A-II and B-II. The 16 new sequences from one isolated location in tropical northern Western Australia all grouped into subgroup B-II, which contained no other isolates. In contrast, the three sequences from the Northern Territory fitted into A-II with 94.6-99.0% nt identities with isolates from the United States, Iran, China and Japan. The 23 new sequences from the central west coast and two east coast locations all fitted into A-I, with 95.9-98.9% nt identities to sequences from Europe and Japan. These findings suggest that (i) there have been at least three separate ZYMV introductions into Australia and (ii) there are few changes to local isolate CP sequences following their establishment in remote growing areas. Isolates from A-I and B-II induced chlorotic symptoms in inoculated leaves of Chenopodium quinoa, but an isolate from A-II caused symptomless infection. One of three commercial ZYMV-specific antibodies did not detect all Australian isolates reliably by ELISA. A multiplex real-time PCR using dual-labelled probes was developed, which distinguished between Australian ZYMV isolates belonging to phylogenetic groups A-I, A-II and B-II.

Molecular Genetic Characterization of Olpidium virulentus Isolates Associated with Big-Vein Diseased Lettuce Plants.

Lettuce plants showing symptoms of lettuce big-vein disease were collected from fields in the Perth Metropolitan region of southwest Australia. When root extracts from each plant were tested by polymerase chain reaction (PCR) using primers specific to the rDNA internal transcribed spacer (ITS) region of Olpidium brassicae and O. virulentus, only O. virulentus was detected in each of them. The nucleotide sequences of the complete rDNA ITS regions of isolates from five of the root samples and 10 isolates of O. virulentus from Europe and Japan showed 97.9 to 100% identities. However, with the six O. brassicae isolates, their identities were only 76.9 to 79.4%. On phylogenetic analysis of the complete rDNA-ITS region sequences of the five Australian isolates and 10 others, the Australian isolates fitted within two clades of O. virulentus (I and II), and within clade I into two of its four subclades (Ia and Id). Japanese isolates had greatest sequence diversity fitting into both clades and into all of clade I subclades except Ib, while European isolates were restricted to subclades Ib and Id. When the partial rDNA-ITS region sequences of two additional southwest Australian isolates, four from Europe, and four from the Americas were included in the analyses, the Australian isolates were within O. virulentus subclades Ia and Id, the European isolates within subclade Ic, and the American isolates within subclades Ia and Ib. These findings suggest that there may have been at least three separate introductions of O. virulentus into the isolated Australian continent since plant cultivation was introduced following its colonization by Europeans. They also have implications regarding numbers of different introductions to other isolated regions. Lettuce big-vein associated virus and Mirafiori lettuce big-vein virus were both detected when symptomatic lettuce leaf tissue samples corresponding to the root samples from southwest Australia were tested using virus-specific primers in reverse transcription-PCR, so presence of both viruses was associated with O. virulentus occurrence.

Global status of tospovirus epidemics in diverse cropping systems: successes achieved and challenges ahead.

The diseases caused by thrips-transmitted tospoviruses (genus Tospovirus, family Bunyaviridae) are a major constraint to production of important vegetable, legume and ornamental crops in different parts of the world. Tospoviruses are characterized by having tripartite RNA genomes and utilizing both negative and ambisense genome expression strategies. Their often wide and overlapping host ranges, emergence of resistance-breaking strains, circulative and propagative relationship with polyphagous thrips vectors, and difficulties in predicting their outbreaks pose challenges to development and implementation of effective management programmes. Despite these challenges, for a few tospoviruses, considerable progress has been made in successful development and deployment of practical and effective integrated disease management programmes. This has been due to increased understanding of their molecular biology, plant-virus and virus-vector interactions and epidemiology, and to identification of risk factors that contribute to increased disease incidence and of tactics to mitigate those risk factors. However, challenges remain as resistance-breaking or other new strains of known tospoviruses and completely new tospovirus species continue to be described from various parts of the world and have the potential to cause damaging epidemics. To protect crops from the losses caused by severe tospovirus outbreaks, continued vigilance is required to identify and characterize these emerging tospoviruses, determine their impact on crop production, understand their epidemiologies and develop, evaluate and implement control measures to reduce their impact on crop production.

Role of recombination in the evolution of host specialization within bean yellow mosaic virus.

Seven complete genomes and 64 coat protein gene sequences belonging to Bean yellow mosaic virus (BYMV) isolates from different continents were examined for evidence of genetic recombination using six different recombination-detection programs. In the seven complete genomes and a single complete genome of the related virus Clover yellow vein virus (ClYVV), evidence for eight recombination patterns was found by four or more programs, giving firm evidence of their presence, and five additional recombination patterns were detected by three or fewer programs, giving tentative evidence of their occurrence. When the nucleotide sequences of 64 BYMV and one ClYVV coat protein genes were analyzed, three firm recombination patterns were detected in 21 isolates (32%). With another six isolates (9%), tentative evidence was found for three further recombination patterns. Of the 19 firm or tentative recombination patterns detected within and between strain groups of BYMV, and with ClYVV, 12 involved a generalist group of isolates as a parent but none of the other BYMV groups acted as parents more than six times. These findings suggest that recombination played an important role in the evolution of BYMV strain groups that specialize in infecting particular groups of domesticated plants.

Quantifying effects of seedborne inoculum on virus spread, yield losses, and seed infection in the pea seed-borne mosaic virus-field pea pathosystem.

Field experiments examined the effects of sowing field pea seed with different amounts of infection with Pea seed-borne mosaic virus (PSbMV) on virus spread, seed yield, and infection levels in harvested seed. Plots were sown with seed with actual or simulated seed transmission rates of 0.3 to 6.5% (2005) or 0.1 to 8% (2006), and spread was by naturally occurring migrant aphids. Plants with symptoms and incidence increased with the amount of primary inoculum present. When final incidence reached 97 to 98% (2005) and 36% (2006) in plots sown with 6.5 to 8% infected seed, yield losses of 18 to 25% (2005) and 13% (2006) resulted. When incidence reached 48 to 76% in plots sown with 1.1-2 to 2% initial infection, seed yield losses were 15 to 21% (2005). Diminished seed weight and seed number both contributed to the yield losses. When the 2005 data for the relationships between percent incidence and yield or yield gaps were plotted, 81 to 84% of the variation was explained by final incidence and, for each 1% increase, there was a yield decline of 7.7 to 8.2 kg/ha. Seed transmission rates in harvested seed were mostly greater than those in the seed sown when climatic conditions favored early virus spread (1 to 17% in 2005) but smaller when they did not (0.2 to 2% in 2006). In 2007, sowing infected seed at high seeding rate with straw mulch and regular insecticide application resulted in slower spread and smaller seed infection than sowing at standard seeding rate without straw mulch or insecticide. When data for the relationship between final percent incidence and seed transmission in harvested seed were plotted (all experiments), 95 to 99% of the variation was explained by PSbMV incidence. A threshold value of <0.5% seed infection was established for sowing in high-risk zones.

Molecular Characterization of Sweet potato feathery mottle virus (SPFMV) Isolates from Easter Island, French Polynesia, New Zealand, and Southern Africa.

Strains of Sweet potato feathery mottle virus (SPFMV; Potyvirus; Potyviridae) infecting sweet-potato (Ipomoea batatas) in Oceania, one of the worlds' earliest sweetpotato-growing areas, and in southern Africa were isolated and characterized phylogenetically by analysis of the coat protein (CP) encoding sequences. Sweetpotato plants from Easter Island were co-infected with SPFMV strains C and EA. The EA strain isolates from this isolated location were related phylogenetically to those from Peru and East Africa. Sweetpotato plants from French Polynesia (Tahiti, Tubuai, and Moorea) were co-infected with SPFMV strains C, O, and RC in different combinations, whereas strains C and RC were detected in New Zealand. Sweetpotato plants from Zimbabwe were infected with strains C and EA and those from Cape Town, South Africa, with strains C, O, and RC. Co-infections with SPFMV strains and Sweet potato virus G (Potyvirus) were common and, additionally, Sweet potato chlorotic fleck virus (Carlavirus) was detected in a sample from Tahiti. Taken together, occurrence of different SPFMV strains was established for the first time in Easter Island, French Polynesia, and New Zealand, and new strains were detected in Zimbabwe and the southernmost part of South Africa. These results from the Southern hemisphere reflect the anticipated global distribution of strains C, O, and RC but reveal a wider distribution of strain EA than was known previously.

An epidemiological model for externally sourced vector-borne viruses applied to Bean yellow mosaic virus in lupin crops in a Mediterranean-type environment.

A hybrid mechanistic/statistical model was developed to predict vector activity and epidemics of vector-borne viruses spreading from external virus sources to an adjacent crop. The pathosystem tested was Bean yellow mosaic virus (BYMV) spreading from annually self-regenerating, legume-based pastures to adjacent crops of narrow-leafed lupin (Lupinus angustifolius) in the winter-spring growing season in a region with a Mediterranean-type environment where the virus persists over summer within dormant seed of annual clovers. The model uses a combination of daily rainfall and mean temperature during late summer and early fall to drive aphid population increase, migration of aphids from pasture to lupin crops, and the spread of BYMV. The model predicted time of arrival of aphid vectors and resulting BYMV spread successfully for seven of eight datasets from 2 years of field observations at four sites representing different rainfall and geographic zones of the southwestern Australian grainbelt. Sensitivity analysis was performed to determine the relative importance of the main parameters that describe the pathosystem. The hybrid mechanistic/statistical approach used created a flexible analytical tool for vector-mediated plant pathosystems that made useful predictions even when field data were not available for some components of the system.